This proposal will investigate Epstein - Barr virus (EBV) as a cofactor in the pathogenesis of a growing subset of HPV+ oropharyngeal squamous cell carcinomas (OSCC). High risk HPV cause epithelial tumors of the cervix, anogenital tract and oropharynx, with the latter rising at epidemic rates. An increased incidence of HPV+OSCC is also observed in HIV-infected individuals. Although the etiology of cervical lesions is well understood, the development of HPV+ OSCC, predominantly arising from the tonsils and base of tongue (BOT), is not. No precursor lesions have been found in HPV-associated OSCC. Tumors develop rapidly with patients presenting with highly metastatic tumors; yet tumors are more responsive to treatment. These biological traits of OSCC differ from those observed for cervical cancers and raise the possibility for other cofactors in the etiology of HPV+ OSCC. The majority of HPV-associated oral lesions arise from the tonsils and BOT which are in close proximity to lymphoid tissues, sites where EBV persists in greater than 90% of the adult population. Indeed, we have observed that a significant number of HPV-positive BOT and tonsillar carcinomas carry EBV, suggesting that EBV may contribute to the development of OSCC. Our data indicate that EBV is able to epigenetically reprogram infected epithelial cells with characteristics that involve CpG island hypermethylation, altered differentiation, increased cell invasion, and activated Wnt signaling. Based on these observations, we hypothesize that EBV infection epigenetically reprograms epithelial cells to favor HPV pathogenesis and the metastatic phenotype observed in HPV+OSCC. Mechanistically, we propose that the EBV-induced epigenetic changes functionally replace the accumulation of mutations typically associated with the progression of HPV+ cervical cancers and provide an explanation for the rapid progression of HPV+OSCC. To test our hypothesis, experiments in aim 1 will define epigenetic alterations induced by EBV infection in non-transformed orally derived epithelial cell lines that regulate epithelial differentiation an invasion. Aim 2 will identify viral and cellular transcriptional signatures of EBV+ and HPV+ OSCC tumors associated with differentiation and invasion. Aim 3 will determine the epigenetic outcomes of EBV and HPV co-infection in organotypic raft cultures. Our studies will provide a mechanistic explanation for the distinct etiology of HPV-associated OSCC. Genome-wide maps of DNA methylation associated with EBV infection will provide a framework for EBV's contribution to the progression of oral carcinogenesis. Using biological relevant in vitro models, we will determine how EBV and HPV interact to accelerate transformation processes and increase invasiveness. Finally, we will identify a set of virally mediated alterations that can sere as biomarkers for detection, prognostic indicators for disease progression, and as therapeutic targets of HPV+ and EBV+ oral cancers including those that arise in the context of HIV/AIDS.